Journal of Fisheries and Aquatic Science
OPEN ACCESS
ISSN 1816-4927
DOI: 10.3923/jfas.2016.
Research Article
It is all in the Blood: Erythrocyte Characterization of Triploid and
Diploid African Catfish, Clarias gariepinus
1
Normala Jalil, 1Mohd Azizul Alim,
1,2
Shahreza Md Sheriff
1,2
Ambok Bolong Abol-Munafi,
1,2
Nur Asma Ariffin, 2Khor Waiho and
1
School of Fisheries and Aquaculture Sciences, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia
Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia
2
Abstract
Objective: A study was done to characterize the erythrocyte of triploid African catfish. Methodology: Triploid African catfish Clarias
gariepinus fingerlings were produced via cold shock method and their erythrocyte profiles were compared with normal diploid controls.
Results: Approximately 99% of triploid erythrocytes were ellipsoidal whereas 97.5% of the diploid erythrocytes were round in shape. All
of the erythrocyte parameters showed significant higher values in triploids and exhibited size range exclusive to triploids except
erythrocyte cellular volume. The nuclear volume and nuclear area were among the two erythrocyte parameters that showed the largest
increment in triploids (76.6 and 55.3%, respectively) with their respective exclusive triploid range at 74.3-150.2 and 22.8-35.2 :m. Both
erythrocyte and nuclear major axis increased at a much higher percentage with respect to erythrocyte and nuclear minor axis
in triploid specimens, resulting in the ellipsoidal shape of triploid s erythrocytes. The erythrocyte major axis also has the highest
percentage (76.0%) of erythrocytes that fall in the exclusive triploid range (11.9-14.9 :m) compared to the other erythrocyte parameters.
Conclusion: Therefore, it is suggested that determination of triploid African catfish can be performed solely based on the measurement
of erythrocyte major axis.
Key words: African catfish, aquaculture, characterization Clarias gariepinus, erythrocyte, triploid
Received:
Accepted:
Published:
Citation: Normala Jalil, Mohd Azizul Alim, Ambok Bolong Abol-Munafi, Nur Asma Ariffin, Khor Waiho and Shahreza Md Sheriff, 2016. It is all in the blood:
Erythrocyte characterization of triploid and diploid african catfish, Clarias gariepinus. J. Fish. Aquat. Sci., CC: CC-CC.
Corresponding Author: Shahreza Md Sheriff, Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu,
Malaysia Tel/Fax: (+60) 09-6683503/(+60) 09-6683390
Copyright: © 2016 Normala Jalil et al. This is an open access article distributed under the terms of the creative commons attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Competing Interest: The authors have declared that no competing interest exists.
Data Availability: All relevant data are within the paper and its supporting information files.
J. Fish. Aquat. Sci., 2016
INTRODUCTION
MATERIALS AND METHODS
Triploidy is the genomic state of having three complete
sets of chromosomes and it is relatively easy to be induced in
fish. Triploid animals are produced by inhibiting the release of
the first or second polar body through the application of a
chemical or environmental stress soon after fertilization1.
Triploid fish do rarely occur in nature and they can be induced
as well. Most of triploid inductions were done using cold shock
treatments2-5 and heat shock treatments6,7. Triploids have been
successfully produced for many commercially important fish
species8-11 and shellfish12-15.
Methods such as erythrocyte measurement, karyotyping
chromosome, flow cytometry, microfluorometry and
Nucleolar Origin Region (NOR) have been used to determine
the triploidy status in organisms. Karyotyping chromosome
method is a time-consuming and tedious method that
estimates triploid chromosomes in an organism, requires the
use of cytotoxic chemical and colchicines to interrupt the
mitotic division to give readable chromosome spreads16.
Meanwhile, flow cytometry and microfluorometry are
laser-based and fluorescent-based methods capable of
estimating the number of triploid cell nuclei, but involve
costly and specialized equipments17-19. From all the methods,
erythrocyte measurement is the easiest, quickest and
cost-effective method to determine triploidy in an organism
without sacrificing the fish and is suitable for
commercialization of triploid fishes20.
Several studies have shown that the size of erythrocytes
in triploid organisms are generally larger compared to
diploid s21-23. However, previous study has reported that the
distribution size of erythrocyte can overlap between triploid
and diploid specimens24. This has risen as an issue as
determination of triploidy in an organism based on
erythrocyte measurement can be inaccurate. Due to this,
detailed characterization of triploid erythrocyte is crucial in
order to understand the actual erythrocyte profile of triploid
organisms. The characterization of erythrocyte profiles based
on species would provide accurate information on triploid
organisms for both researchers and farmers. In addition, it can
also assist in managing triploid organisms during
commercialization as African catfish is an important local fish
species cultured in Malaysia. Currently triploid African catfish
has been successfully produced by several researchers25,26,23 via
cold shock treatments. Thus, this study was done to compare
the erythrocyte characteristics of diploid and triploid African
catfish, Clarias gariepinus and to characterize the erythrocyte
parameters of triploid C. gariepinus for identification of
triploidy in this species.
Broodstocks weighing between 620-920 g were collected
from a local dealer and maintained in one-tonne tank at the
UMT freshwater hatchery until matured and ready to be used
for breeding. Daily feeding using pellet (approximately
3% of b.wt.) was applied. Healthy and matured
broodstocks were injected with GnRH (OVAPRIM, Canada) at
a dosage of 0.5 mL kgG1 for males and 1.0 mL kgG1 for
females27. A minimum acclimatization period of 10 h
post-injection was given for each isolated individuals prior
eggs and sperm collection. Eggs were collected in a clean
bowl by stripping the female softly along her abdomen.
Simultaneously, male was dissected and its sperm sac was cut
open and squeezed to obtain the stored sperms. The collected
eggs and sperms were mixed evenly before subjecting the
fertilized eggs to cold shock method. The cold shock method
was based on baseline parameters set for this species2,28,29.
Triploidy was induced by exposing fertilized eggs at 5EC for
20 min, 3 min after fertilization. Eggs without cold shock
treatment were regarded as control (diploid). Three replicates
were carried out for each triploid and diploid treatment. Each
treatment was maintained separately in 100 L tank with strong
aeration until hatching.
After 2 weeks, 20 fingerlings from each treatment were
randomly selected for erythrocyte characterization. Blood was
obtained by cutting the caudal fin using a pair of surgical
scissors without sacrificing the fish. Triploidy was assessed by
erythrocyte measurement method and slide was prepared
using dry blood smear method21,30. A drop of blood was
dripped on a glass slide and gently smeared using a cover slip.
The smeared blood was air dried for 2 min before fixing with
95% alcohol and air dried again. The slide was then stained
with 10% Giemsa stain for 1 h before washing off the excess
Giemsa stain with distilled water at room temperature. The
Giemsa-stained slide was air dried, mounted with distyrene
plasticizer and xylene (DPX) and sealed with a cover slip. A
compound microscope at 40X magnification (Nikon Eclipse
80i, Japan) was used to measure the cell major axis, cell minor
axis, nucleus major axis and nucleus minor axis (Fig. 1). The
smeared erythrocyte was sectioned into five different blocks
(Fig. 2). Ten erythrocytes were measured for each one block.
A total of 50 erythrocytes were measured for each individual.
Formulae below were used to calculate the cell volume,
nucleus volume, cell area, nucleus area and percentage of
triploid. Data was statistical analyzed by using Independent
t-test:
Volume of erythrocyte = 4/3× π ×(A/2)×(B/2)2
Area of erythrocyte = π ×A×B
2
J. Fish. Aquat. Sci., 2016
triploid and diploid erythrocyte size. This showed that not all
RESULTS
triploid erythrocyte were bigger in size than diploid. However,
Triploid African catfish were successfully produced using
even though there were overlapping in erythrocyte size,
cold shock technique with a success rate of 96-100%.
there were a range of sizes that only occurred in triploid
Approximately 99% of triploid erythrocytes were ellipsoidal
erythrocytes. Triploid s erythrocytes have larger ranges in
while 95.7% of diploid erythrocyte had round shape (Fig. 3, 4).
almost all erythrocyte parameters, except for erythrocyte
axis (12.4±0.7 µm),
volume, even though the lower range limit was still very much
erythrocyte minor axis (8.3±0.7 µm), nucleus major axis
higher in triploid (237.0 :m3) compared to the erythrocyte
(6.0±0.6 µm) and nucleus minor axis (3.3±0.4 µm) were
volume of diploid (114.7 :m3). Excluding erythrocyte volume,
significantly larger (p<0.05) compared to that of diploid s
all other erythrocyte parameters showed an upper exclusive
(Table 1). The erythrocyte area, nucleus area, erythrocyte
triploid range (Table 1). The erythrocyte parameter with the
volume and nucleus volume were significantly larger in
highest percentage of triploid erythrocytes being larger than
triploid specimens as well with an increment of 26.4, 35.6, 31.7
the normal diploid erythrocyte range was erythrocyte major
and 43.4%, respectively. Ratio of diploid:triploid was highest
axis followed by nucleus major axis and nuclear area with a
at erythrocyte nuclear volume (1:1.77) followed by nuclei area
76.0, 54.0 and 21.0%, respectively (Table 1).
Triploid s
erythrocyte
major
(1:1.55) and erythrocyte volume (1:1.46) (Table 1).
DISCUSSION
The increment of one chromosome number in triploid
fishes showed a range size which only exist on triploid fish but
do not exist in diploid fish yet, there is overlapping between
The erythrocyte shape between triploid and diploid varies
because of the increment of one chromosome set in the
triploid fish. The change in erythrocyte shape from round to
ellipse and obvious increase in size in triploid C. gariepinus
A
was observed in triploids of other fish species as well,
a
Oreochromis niloticus31, loach
Misgurnus anguillicaudatus32, wels Silurus ganis33 and shi
drum Umbrina cirrosa34. The increase in overall erythrocyte
such as
b
B
red
tilapia
size was expected as number of chromosome increases in
triploid specimens20,35-38. The change in cell shape and increase
Fig. 1: Sketch of erythrocyte measurement, A: Cell major axis
in cell size is postulated to be a cytoplasmatic adjustment to
of erythrocyte, B: Cell minor axis of erythrocyte,
the increase in nuclear size-enlargement of the cellular
a: Nucleus major axis of erythrocyte and b: Nucleus
major axis more than the cellular minor axis of the triploid s
minor axis of erythrocyte
erythrocytes39,40. This explains the change of erythrocyte shape
Fig. 2: Randomly sampled block design
3
J. Fish. Aquat. Sci., 2016
from round in diploid C. gariepinus to ellipse in triploid
triploid individuals. Similar patterns of increment was
specimens as the erythrocyte major axis increased by 25.3%
reported in caspian salmon Salmo trutta caspius, where the
but the erythrocyte minor axis only increased by 7.8% in
ellipsoidal shape of triploid s erythrocyte is due to the higher
increment of erythrocyte major axis 27% compared to the 22%
increment of the erythrocyte minor axis41.
Triploid
Diploid
The results from this study clearly demonstrated that
erythrocyte parameters such as the nuclear and cellular area
99
97.5
and volume were significantly higher in triploid C. gariepinus
than its diploid counterparts. Several studies have shown that
triploid samples can be distinguished by observing the nuclear
volume which is typically 1.5 times larger than normal42-44. The
4.3
triploid erythrocyte nuclear volume of C. gariepinus in this
0.5
study was 1.77 times larger than diploid s erythrocyte nuclear
volume, similar to that found in red tilapia O. niloticus
Oval (%)
Rounded (%)
(1.68x larger in triploid) by Jayaprasad et al.31 and thai silver
barb Puntius gonionotus (1.63x larger in triploid) by
Koedprang and Na-Nakron45. These are still usual compared to
Fig. 3: Percentage of erythrocyte shape (Oval or rounded)
the erythrocyte nuclear volume of triploid stinging catfish,
between diploid and triploid
(b)
(a)
Fig. 4(a-b): Erythrocyte of triploid and diploid, (a) Triploid erythrocyte more ellipsoidal and larger than diploid and
(b) Diploid erythrocytes more rounded compared to triploid s
Table 1: Comparison of range size, mean and ratio of erythrocyte between triploid and diploid African catfish (Clarias gariepinus) sample (n = 100)
Exclusive
Percentage
Percentage
of triploid
of triploid
Range (:m)
Mean (:m)
Ratio
erythrocyte
-----------------------------
----------------------------
(Diploid:
Increment triploid
erythrocyte
in exclusive
in overlapping
Erythrocyte parameters
Diploid
Triploid
Diploid
Triploid
triploid)
(%)
Erythrocyte major axis (:m)*
7.8-11.8
10.9-14.9
9.9±0.7
12.4±0.7
1:1.25
25.3
11.9-14.9
76.0
Erythrocyte minor axis (:m)*
4.8-10.7
6.1-10.6
7.7±0.7
8.3±0.7
1:1.08
7.8
10.3-10.6
0.3
99.7
Erythrocyte cellular area (:m2)*
35.8-92.1
56.2-108.1
59.9±7.5
81.4±8.0
1:1.36
35.9
92.2-108.1
12.0
88.0
range (:m) triploid range
range
24.0
Erythrocyte cellular volume (:m3)* 114.7-619.8 237.0-619.3
311.0±65.9 455.2±78.6
1:1.46
46.4
-
0.0
100.0
Nucleus Major Axis (:m)*
3.0-6.2
3.3-6.9
4.4±0.5
6.0 ±0.6
1:1.36
36.4
6.3-6.9
54.0
45.0
Nucleus Minor Axis (:m)*
2.1-4.9
2.1-6.4
3.3±0.4
3.8±0.5
1:1.15
15.2
5.0-6.4
3.0
97.0
Nuclear Area (:m2)
5.5-22.7
7.6-35.2
11.4±2.1
17.7±3.4
1:1.55
55.3
22.8-35.2
21.0
79.0
Nuclear Volume (:m3)*
8.1-74.2
11.6-150.2
25.6±7.4
45.2±14.3
1:1.77
76.6
74.3-150.2
10.0
90.0
*Parameters with significant difference (p<0.05)
4
J. Fish. Aquat. Sci., 2016
Heteropneustes fossilis which was 2.28 times increment in
50 erythrocytes for each triploid fish compared to only
24
triploids instead from usual 1.5 times for other species . The
diploid:triploid ratio of erythrocyte nuclear in C. gariepinus
(1:1.55) was consistent with the diploid:triploid ratio
found in other fish species such as 1:1.50 in red tilapia
O. niloticus31, 1:1.56 in wels S. glanis33 and 1:1.50 in sea bass
Dicentrarchus labrax3.
Several studies have stated that the measurement on
major axis is the most accurate and variable value for the
differentiation of triploid and diploid individuals as triploidy
affected the erythrocyte major axis more than the minor
axis2,46,21,11. In this study, erythrocyte major axis has the highest
percentage of triploid erythrocyte (76%) in the exclusive
triploid range. Thus, in order to reduce determination time,
solely based on erythrocyte major axis parameter is sufficiently
effective to determine ploidy level in African catfish.
Similar observations were also reported by Peruzzi et al.47 and
Gheyas et al.24.
The overlapping in size of triploid erythrocyte characters
could be due to biological factors. In blood, the shape and size
of erythrocytes varies as they progress from immature to
mature stages. Studies showed that immature erythrocyte is
reported to be rounder while the nucleus is more centrally
located as compared to mature erythrocyte48. Thus, overlaps
in erythrocyte size between triploid and diploid fishes were
expected, as seen in this study.
Although, previous researches agree that the erythrocyte
size of triploid African catfish fish is larger than the diploid21-23,
no report has yet been published on the percentage of
overlapping between diploid and triploid erythrocyte size and
percentage of erythrocyte sizes in triploid that is larger than
diploid erythrocyte. This is the first report on the
characteristics of triploid African catfish erythrocyte and also
on triploid fish. It is suggested to observe the differences
through the percentage of overlaps and the range of size
present only in triploid individuals. For example, the size range
between 11.9 µm until 14.9 µm of erythrocyte major axis only
exists in triploid individuals (Table 1). These exclusive triploid
ranges in erythrocyte parameters serve as a baseline for future
ploidy determination in African catfish, C. gariepinus.
10 erythrocytes used by Karami et al.23 for analysis. Besides
that, the size of erythrocyte changes in different life cycle. The
average erythrocyte size in the fingerling of Atlantic salmon,
Salmo salar was 101.13 µm2 and for adult fish, the
erythrocytes was slightly larger49, 103.12 µm2.
In fish, red blood cells have shown differences in shape if
taken from different parts of the body and at different ages.
Nilsson et al.50 showed that the erythrocyte became more
elongated when squeezed between pillar cells of gills. They
observed that in rainbow trout Oncorhynchus mykiss and
roach Rutilus rutilus where the erythrocytes of both fish
species were ellipse in the arterioles and arteries. During the
erythrocyte passage through the gill lamella, the mean
erythrocyte length was increased to 18.4±2.1 :m for trout
and to 18.1±2.7 :m for roach. Similar observation was also
noted in an earlier histological observation of fish secondary
lamella51. Besides that, the change in erythrocyte size can
occur in other part of fish body51. In this study, all fish blood
was taken from the caudal fin and at the same age to ensure
uniform conditions.
CONCLUSION
This study successfully highlights the differences between
triploid and diploid African catfish based on erythrocyte
measurement. The erythrocyte major axis is the most suitable
parameter to differentiate between triploid and diploid African
catfish as approximately 76% of the triploid erythrocytes were
in the exclusive triploid range (minimal overlapping with
diploid s range) for this parameter. The usage of erythrocyte
measurement method in determining triploid African catfish
culture will benefits researchers and aqua-culturists as it is
rapid, accurate and does not require sacrificing of fish samples.
ACKNOWLEDGMENT
The researchers wish to thank School of Fisheries and
Aquaculture Sciences, Universiti Malaysia Terengganu,
Therefore, this study demonstrates the potential of using
Terengganu, Malaysia for providing the study facilities.
erythrocyte measurement method to accurately distinguish
between triploid and diploid African catfish. There might
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